IV. Microbiology of Meat Curing A Lyophilized Pediococcus cerevisiae Starter Culture for Fermented Sausage' R. H. DEIBEL, G. D. WILSON, AND C. F. NIVEN, JR. Divisions of Bacteriology and Food Technology, American Meat Institute Foundation, The University of Chicago, Chicago, Illinois In a previous study (Deibel, Niven, and Wilson, 1961) some microbiological aspects of fermented sausage manufacture were investigated with the ultimate aim of employing a starter culture to effect greater control in the production of these meat items. In the study a trend was noted in the industry to eliminate the necessity for a microbial reduction of nitrate by employing a nitritecontaining cure. Thus, one of two microbial-mediated reactions was circumvented; however, chance contamination by fermentative-type bacteria was practiced characteristically. As a consequence of these observations attention was focused on the sugar fermentation effected by the contaminating lactic acid bacteria. In the present report the factors influencing the choice of the starter culture, some factors influencing its preparation for distribution, and its utilization under commercial conditions are described. MATERIALS AND METHODS Culture methods. Cultures were maintained by daily transfer in APT broth2 (Evans and Niven, 1951). Lactobacilli were incubated at 30 C and pediococci at 37 C. Large volumes of cultures used for the inoculation of sausage were grown in APT broth for 24 hr, the cell crop harvested by centrifugation, resuspended in distilled water, and sprayed directly onto the sausage mixes. Lyophilized cultures were prepared by resuspending the cell crops in reconstituted skim milk powder, rapidly freezing in an ethanol-dry Ice mixture, and drying in vacuo over phosphorus pentoxide for 4 to 5 hr. A method was developed for the quantitative estimation of pediococci in which decimal dilutions of the blended sausage samples were plated in the Lactobacillus selective medium3 of Rogosa et al. (1953) and incubated at 45 C for 48 hr. As noted in previous studies only an occasional Lactobacillus strain from meat sources was capable of growth at 45 C. In addition, this medium effectively inhibits the growth of enterococci. Thus, this ' Journal paper no. 207, American Meat Institute Foundation. 2 APT medium contains per L: tryptone, 10 g; yeast extract, 5 g; NaCl, 5 g; K2HP04, 5 g; sodium citrate, 5 g; glucose, 10 g; Tween 80, 1 g; MgSO4-7H20, 0.8 g; MnCl2v4H20, 0.14 g; and FeS04-7H2O, 0.04 g. For plating, add 1.5 per cent agar. 3Baltimore Biological Laboratory, Inc., Baltimore, Maryland. Received for publication September 6, 1960 239 combination of environmental factors proved to be very effective for the quantitative estimation of high acid producing pediococci in the inoculated Pilot plant studies. The formulation and inoculation of the sausage mixes was conducted in an experimental sausage kitchen maintained at 45 to 50 F. After the salt, sugar, spice, and nitrite were added to the meat components, the inoculum was sprayed onto the sausage mixture (table 1) toward the end of the mixing operation. Uninoculated batches were always prepared before inoculated batches to minimize contamination. When two or more strains were tested, all equipment was dismantled, scrubbed thoroughly with soap and hot water, and reassembled between the preparation of each batch. The sausage mixes were stuffed into 3.5 in. diameter cellulose casings and placed in an air-conditioned smokehouse. Generally, the smokehouse temperature was maintained at 80 to 90 F and 90 per cent relative humidity for the first 12 to 16 hr. For the next 24 hr smoke was applied, the temperature was raised to 100 F, and the relative humidity lowered to 80 or 85 per cent. In the last 4 to 8 hr of processing, the temperature was increased gradually until the product attained an internal temperature of 137 F. After removal from the smokehouse, the sausage was held for 48 to 72 hr at 50 F prior to organoleptic evaluation. Characteristically, freshly processed sausage of this variety has a sharp, acid taste and requires at least 48 to 72 hr to mellow. Commercially, this phase of man- TABLE 1 A typical summer-sausage formula employed in pilot plant studies Component Per Cent Trimmed cow rounds 33.3 Trimmed bull rounds 33.3 Pork trimmings.. 28.5 Black pepper.. 0.42 Nutmeg 0.05 Coriander.. 0.12 Glucose... 1.4 Sodium chloride.. 2.9 Sodium nitrite 0.01
'240 R. H. DEIBEL, G. D. WILSON, AND C. F. NIVEN, JR. [VOL. 9 ufacture takes place in the plant or in transit to the consumer. EXPERIMENTAL RESULTS Pilot plant studies with freshly harvested cell crops. In initial experiments, sausage batches were prepared with various Pediococcus and Lactobacillus strains that were harvested from the growth medium immediately prior to inoculation. These sausages always possessed a superior texture in comparison to the soft, uninoculated control No vast differences in the texture or organoleptic properties of the sausages inoculated with the various lactic acid bacteria could be detected. Organoleptically, the uninoculated batches often could be characterized by their lack of tangy flavor as well as an unclean or cheeselike flavor. Generally, no differences in color could be detected in the final products although color deveiopment in the control batches was slower during the smoking process. After preliminary experiments and in consideration of results obtained from the examination of commercial emulsions, the conventional holding period of the sausage at 45 F for 3 days prior to smoking was eliminated in the pilot plant studies. This change did not alter any properties of the inoculated products in any detectable manner. As observed previously (Deibel et al., 1961) no microbial activity was detected during this period. Thus, in the manufacture of summer sausage and its varieties, these considerations cast considerable doubt on the necessity of this phase of sausage manufacture. The final ph values of the inoculated sausages were 4.7 to 5.1 in contrast to uninoculated samples with a range 5.0 to 5.5. Although the respective absolute values would vary from batch to batch, invariably the ph values of the inoculated sausages would decrease more rapidly and attain a lower value than the uninoculated Experiments concerned with methods of distribution and inoculation. In the cheese industry where the employment of starter cultures has been well established, the starter culture is maintained by regular subcultures in small volumes of milk. When needed, larger volumes of starter cultures are prepared in milk from the smaller inocula, which are then used to seed the vats of milk. Such a procedure did not appear to be readily adaptable to sausage making and it was considered that a lyophilized culture, capable of reconstitution and direct addition to the sausage emulsion, would be the ideal method of inoculation. This method offered obvious advantages over the distribution of a cell paste, or cake of cells, or an in-the-plant transfer of the culture. Representative Lactobacillus and Pediococcus strains were lyophilized as described previously. The results with lyophilized Lactobacillus strains were disappointing as high viable cell recoveries were not obtained with any of the strains. However, the Pediococcus strains were found to be resistant to the Iyophilization process. As determined by plate counts in APT agar, 80 to 105 per cent of the cells survived lyophilization. Due to its rapid growth and resistance to lyophilization, Pediococcus cerevisiae strain FP1 was chosen for further study. This strain was used in all subsequent studies and was ultimately employed as the starter culture for fermented sausage. A discussion of its physiological characteristics and taxonomic position is presented later. Studies were then conducted in the pilot plant with lyophilized preparations of P. cerevisiae strain FP1. Contrary to expectations the preparations completely lacked fermentative ability. Although the Pediococcus starter culture could be quantitatively recovered from the sausage mixes immediately following inoculation, after 12 to 16 hr incubation in the smokehouse they were undetectable. Another Pediococcus strain reacted in a similar manner. Activity testing of lyophilized preparations. To facilitate testing a large number of experimentally lyophilized cultures a laboratory scale sausage test was devised. Ground beef 100 g, with glucose, 1.5 g, sodium chloride, 3.0 g, and sodium nitrite, 0.01 g, was used to simulate a commercial sausage mixture. After inoculation, 100 g of the mix was placed in a 100-ml beaker, covered to prevent evaporation, and incubated at 37 C. In an experiment to determine the feasibility of this method, the cell crop from 1 ml of APT medium was used to inoculate 100 g of the mixture. Uninoculated batches were prepared prior to the test batches. Within 18 to 20 hr the inoculated samples possessed the resilient texture associated with fermented sausage. The ph values of these preparations were in the range of 4.8 to 5.0 and the characteristic cured meat color developed fully. In contrast the uninoculated samples possessed an inferior color, a mushy texture, and a ph value of 5.6 to 6.1. This method of testing the fermentative activity of the starter culture was empioyed in the remainder of this investigation. Using this method, experiments were performed to determine the factor(s) inhibiting the growth of the lyophilized starter culture. Sodium chloride and sodium nitrite were employed as variables and a series of the laboratory meat mixtures were prepared accordingly. A lyophilized preparation containing 90 per cent viable cells was tested. In addition, an uninoculated mixture and a mixture inoculated with a freshly harvested cell crop were included for control purposes. The lyophilized cell preparation evidenced strong fermentative ability in the formulations in which the sodium chloride was omitted. However, the freshly harvested inoculum was active under all conditions regardless of the presence or absence of sodium chloride in the mixture. These observations afforded an explanation of the perplexing results in which high viable cell counts were observed in lyophilized preparations plated in APT agar, and their inactivity in sausage emulsions. Sub-
P. CEREVISIAE STARTER CULTURE FOR FERMENTED SAUSAGE stantiating evidence was obtained when lyophilized preparations were plated in APT agar and APT agar containing 6.0 per cent added sodium chloride. Small numbers of cells (less than 101 per ml of original medium) were able to initiate growth in the salt-containing medium. In contrast, viable counts (per ml of original medium) in the range of 108 to 101 were obtained in the APT agar. Thus, it would appear that the lyophilization process destroyed or interfered with the ability of the organism to tolerate high salt concentrations. In further experiments it was demonstrated that the loss of sodium chloride tolerance in lyophilized preparations could be averted if the inoculum culture was maintained by daily transfer in 6.5 per cent sodium chloride broth. Using this procedure fermentative activity was achieved in the lyophilized preparations and viable plate counts on APT agar and APT agar containiiig 6.5 per cent sodium chloride were comparable. The lyophilized starter culture was then tested under commercial conditions of sausage manufacture. The only restrictions in the formulation of product that were imposea' on cooperating manufacturers consisted of limiting the sodium nitrite concentration in the sausage to a...... ',,',',',,',',,,,,' -.:%-,..':':.,... -------.------"'-'-'- '",. :'... -f,',_i-a---------.!...w: li!,,..,.i,,..i,..!!.i:!.::i. '.."':'' "':i::i:i..,.i.!..--'i. ::"''.--!.i-'.:: ::::":":,:,.:,.. -.::!...--..:i.::!. :'I.1,...:..,.,.,!i -:iii' -:""'"'" --I-----!0..:'..f.. 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Manufactuirers were encouraged to omit the initial holding period of the sausage at 45 F, to place the stuffed sausage directly into the smokehouse, and to employ a 48-hr heating and smoking period. Some manufacturers, whose primary interest was to produce a more uniform prodtuct rather than shortening the processing time, preferred to follow their usual schedules. In preliminary field trials a tendency to overmix the emulsion to insure distribution of the starter culture was observed. This resulted in an excessive blending of the fat and loss of fat-particle definition in the product as well as a somewhat inferior texture. Aside from this minor difficulty a highly acceptable product was produced characteristically. At present, the Pediococcus starter culture4 has re- 4 Under a nonexelusive license with the American Meat Instittute Foundation, Merck & Co., Inc., Rahway, New Jersey, formulate and distribute the Pediococcuts cerezisiae starter ctulture under the trade name of ACCEL. The culture has been approved for use in fermnntel SAiisage by The Meat Inspection Division of TLe UT. S. DLepartment of Agriculture (Miller, 191E6). ir^ :.:s l l- -::::.:4 241 Figure 1. Comparison of the morphology of Pediococcuts cerevisiae strain FP1 when grown in broth culture (left) and sausage (right). The slides, prepared from 20-hr cultures, were stained by the Gram method. Magnification 2300X.
242 R. H. DEIBEL, G. D. WILSON, AND C. F. NIVEN, JR. [VOL. 9 ceived acceptance in the fermented sausage industry. Each commercial batch of the starter culture is thoroughly tested for activity, thus affording greater control over the fermentation and uniform sausage manufacture of high quality. Physiology and taxonomy of the Pediococcus starter culture. Throughout these studies a peculiar morphology of the Pediococcus starter cultures was observed when smears of the sausage were examined. As demonstrated in figure 1, the individual cells were enlarged and lacked the uniformity of cell size associated with cultures grown in the laboratory. Subsequently, this morphology was also observed in smears of cultures grown in 6.5 per cent sodium chloride media. Thus, this peculiar morphology was associated with the growth of the organism in media containing high sodium chloride concentrations. Some physiological characteristics of the Pediococcus starter cultures are presented in table 2. Although this strain was capable of growing at 10 C, its optimal temperature for growth is approximately 37 C. In comparison to the descriptions presented in Bergey's Manual of Dete7minative Bacteriology (Breed, Murray, and Smith, 1957) and by Pederson (1949), it is observed that the starter culture strain possesses characteristics in common with both Pediococcus cerevisiae and TABLE 2 Physiological characteristics of Pediococcus cerevisiae strain FP1 Motility Catalase: APT broth.. 0.05% Glucose CO2 from glucose Nitrate reduction.. Litmus milk.. Final ph, glucose broth Agar shake. Acetylmethylcarbinol production. Hydrolysis: Sodium hippurate Esculin. Arginine. Gelatin. Starch Growth: 10 C.. 30 C.. 45 C.. 50 C.. 5% NaCl, 30 C. 10% NaCl, 30 C No change 3.7 Facultative Xylose, arabinose, mannose, fructose, galactose, sucrose, trehalose, glucose, salicin, cellobiose, and a-methyl-d-glucoside were fermented. Glycerol was fermented only under aerobic conditions. Lactose, maltose, raffinose, inulin, mannitol, melibiose, rhamnose, sorbose, melezitose, dulcitol, dextrin, sorbitol, and a-methyl-d-mannoside were not fermented. Pediococcus acidilactici. The inability to ferment maltose coupled with a high optimal temperature of growth were characteristics of sufficient import to suggest species recognition (Pediococcus linderii) by Shimwell (1948). The starter culture possesses both of these characteristics. Recently, Nakagawa and Kitahara (1959) have extended the taxonomy of this genus and proposed five distinct species. According to their classification scheme, the starter culture shares characteristics in common with two species (Pediococcus acidilactici and Pediococcus pentosaceus). Until a classification scheme is generally accepted, the authors are hesitant to alter species designation of the starter culture although they recognize that this may be necessary sometime in the future. DISCUSSION In the commercial production of fermented foods, manufacturers strive constantly to exercise a high degree of control over the process and produce consistently a uniform product of high quality. In the past, sausage manufacturers were not only beset with large losses of product due to undesired fermentations, but also wide variation in over-all product quality. As has been demonstrated in other fermented food industries the only approach to a controlled fermentation is through the agency of starter cultures. Thus, by the direct addition of the organism producing the desired effect, growth of undesirable bacteria is restricted and uniformity of product is enhanced. Upon introduction of the Pediococcus starter culture, some manufacturers expressed concern over the possible loss of product distinctiveness, specifically organoleptic characteristics, if all manufacturers used the same starter culture. This concern was allayed when the prime factors of organoleptic distinctiveness were considered. Aside from the tangy flavor common to this class of sausages, the predominant organoleptic characteristics arise from the individual formulations in the spices, sugar, salt, and meat components as well as contributions from the varied processing schedules as practiced in the individual plants. Organoleptic contributions due to secondary fermentations (aside from lactic acid production) appear to be of small import in summer-type However, these considerations are not applicable in toto when extended to other varieties of fermented sausage such as Lebanon Bologna or Genoa sausage. In these instances the primary lactic acid fermentation is necessary but secondary fermentations appear to play a significant role in organoleptic distinctiveness of the products. As yet the microbiology of these secondary fermentations has not been elucidated. The employment of starter cultures in the fermented sausage industry was pioneered by Jensen and Paddock (1940). These investigators employed several Lactobacillus species to effect the lactic acid fermentation but de-
1961] P. CEREVISIAE STARTER CULTURE FOR FERMENTED SAUSAGE 243 pended upon chance contamination for nitrate reduction. Conversely, Micrococcus aurantiacus has been used as a starter culture to reduce nitrate (Niinivaara, 1955); however, chance contamination by fermentative bacteria was practiced in this study. With the trend in industry to use nitrite in the cure only the lactic acid fermentation is left to chance. Obviously, this fermentation can be effected by a number of lactic acid bacteria and P. cerevisiae is but one of a large number of these bacteria. Thus, the use of a starter culture on an industrywide basis is then resolved to problems of distribution and in-plant utilization of the culture. The employment of starter cultures by all manufacturers of fermented sausage appears inescapable. A process which affords product uniformity of high quality, relative freedom from failure, and possible savings in time and labor should ultimately replace the traditional process with its hazards resulting from chance contamination. SUMMARY The development of a lyophilized culture of Pediococcus cerevisiae for summer-type fermented sausages stemmed from the necessity of a characteristic lactic acid fermentation, considerations of an industry-wide distribution of the culture, and in-plant inoculation problems. Some difficulty was encountered in the lyophilization of the cells due to the apparent loss of sodium chloride tolerance in the lyophilization process. However, the loss of salt tolerance could be averted by maintaining the culture in a medium containing 6.5 per cent salt. A laboratory method of testing the activity of starter culture preparations is presented. A ground meat mixture approximating the formulation of sausage with regard to curing ingredients, salt, and sugar is inoculated with the preparation in question and incubated. Activity is determined by the acid produced as well as the color and texture of the meat. The starter culture performed satisfactorily under manufacturing conditions and has proved to be useful in the commercial production of a variety of fermented REFERENCES BREED, R. S., MURRAY, E. G. D., AND SMITH, N. R. 1957 Bergey's manual of determinative bacteriology, 7th ed. The Williams & Wilkins Co., Baltimore, Maryland. DEIBEL, R. H., NIVEN, C. F., JR., AND WILSON, G. D. 1961 Microbiology of meat curing. III. Some microbiological and related technological aspects in the manufacture of fermented Appl. Microbiol., 9, 156-161. EVANS, J. B. AND NIVEN, C. F., JR. 1951 Nutrition of the heterofermentative lactobacilli that cause greening of cured meat. J. Bacteriol., 62, 599-603. JENSEN, L. B. AND PADDOCK, L. 1940 Sausage treatment. U. S. patent 2,225,783. MILLER, A. R. 1956 Lactic acid starter culture. U. S. Dept. Agr., Meat Inspect. Div., Memo. No. 234. NAKAGAWA, A. AND KITAHARA, K. 1959 Taxonomic studies on the genus Pediococcus. J. Gen. Appl. Microbiol., 5, 95-126. NIINIVAARA, F. P. 1955 tber den Einfluss von Bakterienreinkulturen auf die Reifung und Umr6tung der Rohwurst. Acta Agral. Fennica, 84, 95-101. PEDERSON, C. S. 1949 The genus Pediococcus. Bacteriol. Revs., 13, 225-232. ROGOSA, M., WISEMAN, R. F., MITCHELL, J. A., DISRAELY, M. N., AND BEAMAN, A. J. 1953 Species differentiation of oral lactobacilli from man including descriptions of Lactobacillus salivarius nov. spec. and Lactobacillus cellobiosus nov. spec. J. Bacteriol., 65, 681-699. SHIMWELL, J. L. 1948 A rational nomenclature for the brewing lactic acid bacteria. J. Inst. Brewing, 54, 100-104